Project Summary Human immunodeficiency virus (HlV-1) is the etiologic agent of acquired immunodeficiency syndrome (AIDS). The global HIV-1 pandemic (~35 million people infected) is sustained by 2-S million new infections annually. Changing the course of this pandemic requires prevention of HlV-1 transmission, most of which occurs sexually. In the absence of an effective vaccine, the use of specific antiretroviral drugs has been investigated and shown to protect, in a partial manner, at-risk sex partners from HIV-1 infection. However, partial efficacy, drug side effects, suboptimal compliance and the emergence of drug-resistant viruses limit the general applicability of these agents as prophylactic measures. New broadly active antiviral agents that can be used as topical microbicides hold the promise of addressing these deficiencies. The HlV-1 envelope glycoproteins (Envs), which mediate virus entry into target cells, represent attractive targets for such prophylactic agents. The HIV-1 Envs are exposed on the viral surface, are accessible to water-soluble inhibitors and are present in low numbers on each virion. Env inhibitors used as microbicides need not be systemically absorbed or taken up by host cells, limiting potential toxicity. Conserved elements of Env mediate receptor binding, conformational changes, and membrane fusion, providing several potential targets for inhibition. Although some HIV-1 entry inhibitors have been identified, drug-resistant HlV-1 variants either exist naturally or develop during treatment. To identify new broad-range inhibitors of HIV-1 entry, we have developed and utilized an Env-dependent cell-cell fusion screening assay. To date, the primary screen has been conducted on more than 600,000 compounds. The hits from the primary screen are in the process of being confirmed and validated, and two novel HlV-1 Env inhibitors have been identified recently. In this project, specific and broad range inhibitors, including analogues modified to improve potency while retaining breadth, will be characterized. We will also identify the target protein, the mechanism of inhibition of HIV-1 entry, and the binding site ofthe inhibitor. The proposed studies should permit us to classify inhibitors into groups; synergy or antagonism among the groups will also be explored. The inhibitors will serve as novel probes to understand the complex, multi-step process of HIV-1 entry, and could represent leads for prophylactic microbicides and treatments for already infected individuals.